Functional probing of the human glucocorticoid receptor steroid-interacting surface by site-directed mutagenesis - Gln-642 plays an important role insteroid recognition and binding

To elucidate which amino acids in the glucocorticoid receptor ligand-bindin g domain might be involved in determining steroid binding specificity by in teraction with the D-ring of glucocorticoids, we have performed site-direct ed mutagenesis of the four amino acids Met-560, Met-639, Gln-642, and Thr-7 39 based on their proximity to the steroid in a model structure. Mutations of these residues affected steroid binding affinity, specificity, and/or st eroid-dependent transactivation. The results indicate that these residues a re located in close proximity to the ligand and appear to play a role in st eroid recognition and/or transactivating sensitivity, possibly by changes i n the steroid-dependent conformational change of this region, resulting in the formation of the AF-2 site. Mutation of Gln-642 resulted in a marked de crease in affinity for steroids containing a 17 alpha-OH group. This effect was alleviated by the presence of a 16 alpha-CH3 group to a varying degree . Thr-739 appears to form a hydrogen bond with the 21-OH group of the stero id, as well as possibly forming hydrophobic interactions with the steroid, Met-EGO and Met-639 appear to form hydrophobic interactions with the D-ring of the steroid, although the nature of these interactions cannot be charac terized in more detail at this point.